Like other RNAs, transfer RNA is transcribed as a precursor and must undergo maturation. RNase P removes the 5'end leader. The endonuclease tRNase Z, a member of the [unreadable]-lactamase family of metal-dependent hydrolases, removes the 3'end trailer so that CCA can be added by tRNA nucleotidyltransferase. 3'end CCA, a tRNase Z anti-determinant, ensures that mature tRNA proceeds smoothly through aminoacylation. Residues in Motif II, a His cluster (HxHxDH) which is the signature sequence of the [unreadable]-lactamase superfamily, are required for tRNase Z catalysis, but not for substrate pre-tRNA binding. Residues in conserved blocks on the carboxy side of Motif II (motifs III, IV, V and the HEAT and HST loops) are involved with metal ion coordination and catalysis but not with substrate binding. Two conserved blocks on the amino side of motif II (the PxKxRN loop and motif I region), on the other hand, are involved with both substrate binding and catalysis, and could contribute to CCA anti-determination. [Aim 1] Previous investigation of the PxKxRN loop and motif I region will be extended by deletion of the PxKxRN loop and by combining mutations in both blocks. [Aim 2] Because the long form of tRNase Z (tRNase ZL) is thought to have originated as a tandem gene duplication of the short form (tRNase ZS), an altered version of the motif I region (?-motif I) may be found toward the amino end of tRNase ZL. We propose to investigate whether ?-motif I participates in tRNase Z catalysis. [Aim 3] Wild type and variant tRNase Zs will be investigated by protease - mass spectroscopy to analyze the exposure of the loops, including the PxKxRN loop, with enzyme free in solution and bound to tRNA. [Aim 4] Naturally occurring mutations in the T loops of human mitochondrial tRNAs are associated with maternally transmitted diseases and syndromes, principally myopathies. Effects of pathogenesis-associated T loop substitutions will be investigated by analyzing structure and tRNase Z processing of wild type and mutant mitochondrial substrates. Experimental procedures, including mutagenesis by overlap-extension PCR, baculovirus expression and affinity purification of mutant tRNase Z, in vitro transcription and labeling to prepare pre-tRNA substrates, Michaelis-Menten analysis of processing kinetics, gel shift determination of Kd for pre-tRNA-tRNase Z complexes, structure probing of pre-tRNAs, and protease - mass spectroscopic analysis to evaluate exposure of loops in the enzyme in the presence and absence of substrate, will lead to detailed insight into the regulation of a biomedically significant enzyme reaction. Additionally, these methods will be instructive to student trainees considering biomedical research careers at the PhD level. PUBLIC HEALTH REVELANCE: Transfer RNA (tRNA) is central to the process of protein synthesis. Mutations in tRNAs are associated with maternally transmitted mitochondrial diseases and syndromes. Additionally, the gene that encodes tRNase ZL (Elac2, one of the enzymes in the tRNA maturation pathway) has been associated with an elevated risk of prostate cancer, making the proposed research biomedically relevant.